Extraction of nucleic acids

Genomic DNA was extracted from cells using the Qiagen QIAamp Blood and Tissue Kit according to manufacturer's specifications.

3.1.3.2 Isolation of mRNA and cDNA synthesis

Total RNA was isolated from MRC5Vi cells, primary WT or XP20BE fibroblasts using the RNeasy Mini Kit by Qiagen. Cells were harvested as described in 3.3.1.1 and disrupted using RLT buffer. Depending on the number of pelleted cells, either 350μl or 600μl RLT buffer was added to the pellet, and further steps were carried out according to the manufacturer's specifications. In order to remove any residual contaminating DNA, DNase treatment was performed. Finally, RNA quantification and its assessment of purity were determined by spectrophotometry using the NanoVuePlus^® (see 3.1.2) and the RNA was stored at -80°C

until further use. Subsequently, cDNA was synthesized from the isolated RNA with the help of the Revert Aid H Minus First Strand cDNA Synthesis Kit. This kit included a reverse transcriptase, an enzyme that uses RNA as a template to synthesize complementary DNA (cDNA) lacking a ribonuclease H activity and therefore, it does not degrade RNA in RNA-DNA hybrids during synthesis of the first strand cRNA-DNA. To synthesize mRNA anchored Oligo (dT)18 primer annealing to the poly-A tail of mRNA molecules was used. In order to generate cDNA, 1µg of total RNA was mixed with 1µl Oligo d(T) primer, aqua bidest was added to 12µl and incubated at 70°C for 5min. In the meantime, a premix consisting of reaction buffer, RNase inhibitor and dNTPs was prepared. The premix was added to the reaction mix and incubated at 37°C for 5min. Thereafter, 1µl reverse transcriptase was added and the cDNA was synthesized at 42°C for 60min. The reaction was stopped by incubation at 70°C for 10min and the synthesized cDNA was stored at -20°C until further use.

3.1.3.3 Alkaline lysis plasmid extraction

Alkaline lysis was first described by Birnboim and Doly in 1979 and has, with a few modifications, been the preferred method for plasmid DNA extraction from bacteria ever since. The procedure starts with the growth of the bacterial cell culture harbouring the plasmid under selective conditions (ampicillin in LB-medium). Cells were pelleted by centrifugation to remove them from the growth medium. The pellet was then resuspended in a solution containing Tris, EDTA, glucose, and RNase A. Bivalent cations (Mg²⁺, Ca²⁺) are essential for DNase activity and the integrity of the bacterial cell wall. EDTA chelates bivalent cations in the solution preventing DNases from damaging the plasmid and also destabilizes the cell wall. Glucose maintains the osmotic pressure so the cells do not burst and RNase A is included to degrade cellular RNA when the cells are lysed. The lysis buffer contains sodium hydroxide (NaOH) and the detergent SDS. SDS is there to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and the plasmid, to single stranded DNA (ssDNA). This denaturation is the central part of the procedure. Addition of potassium acetate (neutralization) decreases the alkalinity of the mixture. Under these conditions the hydrogen bonding between the bases of the single stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA. While it is easy for the small circular plasmid DNA to re-nature it is impossible to properly anneal huge gDNA stretches. As the double-stranded plasmid can dissolve easily in solution, the single stranded genomic DNA, the SDS and the denatured

cellular proteins stick together through hydrophobic interactions to form a white precipitate.

The precipitate can easily be separated from the plasmid DNA solution by centrifugation. The last step is to clean up the solution and concentrate the plasmid DNA. This was performed by binding to glass-silica membranes and ethanol precipitation (see 3.1.1).

For isolation of smaller amounts of plasmid DNA 4ml LB medium containing ampicillin (100µg/ml), according to the selection marker of the plasmid were inoculated shaking overnight at 37°C with a single bacterial colony. According to manufacturer’s instructions of the NucleoSpin^ Plasmid Kit by Machery and Nagel plasmid DNA was isolated. To isolate lager DNA amounts the NucleoBond^® Xtra MiDi/Maxi Kit by Machery and Nagel was used according to the user’s manual. 100ml LB medium containing ampicillin (100µg/ml) were inoculated with bacteria and incubated shaking at 37°C overnight.

3.1.3.4 Agarose gel electrophoresis (AGE)

For analysis or preparative purposes DNA fragments or plasmids, generated by PCR or restriction digestion were subjected to agarose gel electrophoresis. In an electric field negatively charged DNA fragments move towards the anode ((+)-pole). Depending on fragment size DNA fragments exhibit different mobilities in the agarose gel matrix. Smaller fragments move faster than bigger ones so that they can be separated by size and visualized by fluorescence dyes (e.g. HD Green^® Safe DNA Dye) that intercalate into the double helix, and UV-light (254nm). Agarose gels were generated by diluting a suitable amount of agarose powder (e.g. 1%) in 1x TBE buffer and dissolving it by boiling using the microwave. For visualization, a nucleic acid stain (HD Green^® Safe DNA Dye) was added (5µl per 100ml agarose). 1x TBE buffer was used as running buffer at a current between 60-80 V. Samples were prepared with 6x loading buffer and compared to a molecular weight size standard.

Visualization of fragments was performed using the gel documentation system by Intas.

3.1.3.5 Extraction of DNA from agarose gel

Extraction of specific DNA fragments from agarose gels was performed according to the instructions of the NucleoSpin^ Gel extraction and PCR clean up Kit by Machery and Nagel.

Previously, DNA fragments were separated by agarose gel electrophoresis (see 3.1.3.4) and the fragment of interest was excised using a scalpel.

3.1.4 Enzymatic manipulation of DNA